Cigarette smoke (CS) impairs host-protective immune responses to Mycobacterium tuberculosis (MTB). However, an overlooked aspect is how CS or nicotine may drive immunosuppressive cells to increase susceptibility to MTB. T regulatory cells (Tregs) are immunosuppressive cells that predispose laboratory animals and humans to TB. CS or nicotine not only increases the influx of Tregs to the lungs but also induces cell surface signaling molecules ? programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) ? that enhance their immunosuppressive activity. Our central hypothesis is that CS enhances the immunosuppressive function of Tregs via induction of PD-1 and CTLA-4, and that this upregulation of Treg activity plays a pivotal role in impairing control of MTB infection. We will undertake a two-pronged approach, using both an in vivo murine model and an ex vivo model using primary human cells. First, we will adoptively transfer equal number of air- or CS-exposed Tregs into CS-nave mice and then infect the recipient mice with MTB to determine if transfer of CS-exposed Tregs aggravates MTB infection more than transfer of air-exposed Tregs. Second, we will utilize co-cultures of primary human alveolar macrophages (AM) + Tregs with only the Tregs exposed to medium alone, CS, or nicotine, and pharmacologic antagonists to PD-1 and CTLA-4 to establish the mechanism by which CS/nicotine increases Treg activity to impair anti-TB immunity. Aim 1: Establish the role CS-exposed Tregs play in predisposing to MTB in an in vivo murine model. Hypothesis: We predict that CS-induced upregulation of Treg activity is a pivotal mechanism by which CS predisposes to MTB. Thus, compared to mice receiving air-exposed Tregs, mice receiving CS-exposed Tregs will be more susceptible to TB, demonstrated by fewer host-protective M1 macrophages and TH1 cells, greater burden of MTB, worse lung pathology, and reduced survival. Approach: We will adoptively transfer the same number of Tregs from air- or CS-exposed donor mice to CS-nave mice, infect the recipient mice with MTB, and determine microbiological and immunological outcomes. Aim 2: Establish the role CS-exposed Tregs play in impairing human AM control of MTB. Hypothesis: CS or nicotine will increase PD-1 and CTLA-4 expression on Tregs, and in co-cultures with AM, augment the suppressive effect of Tregs on AM, demonstrated by increased levels of immunosuppressive cytokines, decreased levels of host-protective cytokines, reduced phagosome-lysosome (P-L) fusion and autophagy, and greater recovery of MTB. Blocking PD-1 or CTLA-4 will abrogate these effects. Approach: Air-, CS-, or nicotine-exposed Tregs will be co-cultured with nave AM, infected with MTB inhibitors to PD-1 or CTLA-4, and analyzed for MTB burden, cytokines, P-L fusion, and autophagy. This project will clarify the role played by Tregs in mediating CS-induced susceptibility to TB. Findings from these studies can provide the foundation for developing unique immunomodulatory approaches to treat TB.